Two forms of the nonmuscle myosin heavy chain (MHC-A and MHC-B) have been cloned in this laboratory and are the products of two different genes in humans. We would like to understand the function of the MHC-A and MHC-B isoforms. To do this, we wish to over-express myosin fragments from humans in various tissue culture cell lines. Over- expression of the rod fragment, in particular, may contribute to a loss of function because, although the rod can form filaments with intact myosin, it lacks the enzymatic and actin-binding sites located in the head. The human myosin fragments have been subcloned into a mammalian expression vector which has been used to transfect the human melanoma cell line, A2058. A rat basophilic leukemia cell line (RBL-2H3) has also been transfected with the MHC-A and MHC-B rod fragments in the same vector. The cell lysates are positive for the appropriate fragments by size and by binding of antibody on Western blots. We are currently looking at phenotypic changes in cell growth and function. Since the melanoma cell line has previously been used for studies of cell motility, it will be possible to measure the response of transfected and control cells (cells transfected with vector alone or mock-transfected cells). The RBL cells have been well characterized with regard to their secretion of granules in response to antigen stimulation and myosin has been implicated in the release of histamine from these cells. Recent results have shown that RBL cells contain only the MHC-A isoform and so they may be a particularly interesting cell line in which to interfere with myosin function. Antisense constructs will also be used in the same vectors in an effort to disrupt myosin function.